MOLECULAR MEDICINE REPORTS 9: 1681-1688, 2014

Allelic methylation status of CpG islands on 21q in patients with Trisomy 21

YIN‑YIN XIA1, YU‑BING DING2, XUE‑QING LIU2, XUE‑MEI CHEN2, SHU‑QUN CHENG1, LIAN‑BING LI3, MING‑FU MA3, JUN‑LIN HE2 and YING‑XIONG WANG2

Departments of 1Occupational and Environmental Hygiene and 2Reproductive Biology, Chongqing Medical University; 3Chongqing Key Laboratory of Birth Defects and Reproductive Health, Institute for Science and Technology Research of Chongqing Population and Family Planning, Chongqing 400016, P.R. China

Received May 23, 2013; Accepted January 30, 2014

DOI: 10.3892/mmr.2014.1985

Abstract. Trisomy 21 is a chromosomal condition caused by Introduction the presence of all or part of an extra 21st chromosome. There has been limited research into the DNA methylation status Down's syndrome (DS) or trisomy 21 is a chromosomal of CpG islands (CGIs) in trisomy 21, therefore, exploring the condition caused by the presence of all or part of an extra DNA methylation status of CGIs in 21q is essential for the 21st chromosome. DS is a high‑incidence birth defect that development of a series of potential epigenetic biomarkers is often associated with impairment of cognitive ability and for prenatal screening of trisomy 21. First, DNA sequences physical growth (1). Individuals with DS have a higher risk of CGIs in 21q from the USCS database were obtained and of congenital heart disease (CHD), dysfunction of the thyroid 149 sequences and 148 pairs of primers in the BGI YH data- gland, Hirschsprung's disease, eye and hearing disorders, base were aligned. All 300 cases were analyzed by a heavy leukemia and testicular cancer; however, there is a wide range methyl‑polymerase chain reaction (HM‑PCR) assay and a of phenotypic variation in DS (2‑6). It is now more than half a comparison of the DNA methylation status of CGIs was made century since DS was first shown to result from trisomy 21 (7). between trisomy 21 and the control. The HM‑PCR assay Although progress has been made by investigating to results did not show a difference in the DNA methylation status understand the complex phenotypes associated, the mecha- between individuals with trisomy 21 and the control. In total, nisms remain far from clear. there were 11 CGIs that showed various DNA methylation DS disorders are the result of extra copies of the genes statuses between Japanese and Chinese patients. Subsequently, located on . In general, an overexpression bisulfite genomic sequencing found variations in the methyla- of the genes arises and DNA hypomethylation is a possible tion status of CpG dinucleotides in CGIs (nos. 14, 75, 109, 134 mechanism to explain the altered expression. DNA and 146) between trisomy 21 and the control. The different methylation often occurs in a CpG dinucleotide, in which DNA methylation status of CpG dinucleotides in CGIs may the cytosine gains a methyl group. Hypermethylation results be a potential epigenetic marker for diagnosing trisomy 21. in transcriptional silencing, for example genomic imprinting No difference was identified in the DNA methylation status of and X‑chromosome inactivation, while hypomethylation is 21q CGIs among Chinese individuals with trisomy 21 and the linked to chromosomal instability and loss of imprinting. control. The homogeneity of the DNA methylation status of Unmethylated CpGs are often grouped in CpG islands (CGIs), 21q CGIs in Chinese patients indicates that DNA methylation which are present in the 5' regulatory regions of a number of is likely to be an epigenetic marker distinguishing ethnicities. genes and acquire abnormal hypermethylation in numerous disease processes. Alterations of DNA methylation have been recognized as an important component of cancer develop- ment (8). Altered methylation status in peripheral blood lymphocytes (PBLs) has been linked to increased risk of several diseases and, in addition, PBLs are an easily accessible Correspondence to: Dr Jun‑Lin He or Dr Ying‑Xiong Wang, source to identify potential epigenetic biomarkers (9). Department of Reproductive Biology, Chongqing Medical University, East Building, No. 1 Yixueyuan Road, Chongqing 400016, In the present study, a comprehensive CGI methylation P. R. Ch i na analysis was performed using trisomy 21 and control cases to E‑mail: [email protected] identify the allelic methylation status in CGIs of PBLs. The E‑mail: [email protected] aim of the study was to determine the utility of CGI meth- ylation analysis associated with human diseases, in particular, Key words: CpG islands, trisomy 21, DNA methylation, the DS complex phenotypes and to locate potential epigenetic chromosome pair 21, epigenetic marker biomarkers for prenatal diagnosis. To the best of our knowl- edge, there are no comprehensive CGI methylation analyses that have focused on chromosome 21q using a large number 1682 XIA et al: METHYLATION STATUS OF CpG ISLANDS ON CHROMOSOME 21q of trisomy 21 samples (10), thus, the present study focused on Madison, WI, USA) or 100 units McrBC (New England chromosome 21, which is the major contributor of DS complex Biolabs, Ipswich, MA, USA) overnight at 37˚C in 50 µl of phenotype. the buffers recommended by the suppliers. Subsequently, the enzymes were inactivated at 65˚C for 20 min and the levels of Materials and methods digested DNA were determined by electrophoresis. For PCR analysis, 0.5 µl (5 ng) genomic DNA digested with Alignment of CGI data to the BGI YH database. The present each enzyme was used in a 10 µl reaction mixture containing analysis was based on the results of a comprehensive measure- 0.25 units Ex‑Taq DNA polymerase (Takara Bio Inc., Shiga, ment of CGI methylation on human chromosome 21q (11). Japan), 4 nmol dNTP (Takara Bio Inc.) and 10 nmol each Yamada et al repeat‑masked the chromosome sequence and primer [Sangon Biotech (Shanghai) Co., Ltd., Shanghai, computationally identified all non‑repetitive CGIs using China] in 5 µl 2X GC Buffer I or 2X GC Buffer Ⅱ (Takara standard tools and parameters (GC content, >50%; ratio of Bio Inc.). The thermal cycling parameters were recommend observed versus expected number of CpG dinucleotides, >0.6; by Yamada et al (11). The amplified products were electropho- >400 base pairs in length). The authors designed primers for resed on a 2% agarose gel, stained with Goldview nucleic acid the 149 CGI identified and extracted corresponding DNA from stain (SBS Genetech, Beijing, China) and visualized by the samples of human PBLs. Finally, the authors determined the Molecular Imager Gel Doc XR+ System (Bio‑Rad, Hercules, methylation status of each CGI using methylation‑specific CA USA). restriction enzymes (via HpaII‑McrBC‑PCR). The DNA sequences of 149 CGIs were acquired from UCSC (11) and Bisulfite genomic sequencing. Human genomic DNA (2 µg) were aligned with the sequence in BLAST, the BGI YH from PBLs was treated with sodium bisulfite according to the database (http://yh.genomics.org.cn/search.jsp). The score of standard procedure. One‑tenth of the bisulfite‑treated DNA each alignment was indicated by one of five colors, in which was used for PCR in a 50 µl reaction mixture, 10X PCR Buffer the highest score was >200 and shown in red. Multiple align- [100 mM Tris‑HCl (pH 8.8), 500 mM KCl, 15 mM MgCl2 and ments on the same database sequence were connected by a 0.8% (v/v) Nonidet P40], 10 nmol each dNTP, 10 nmol each striped line. A continuous red line indicated a perfect match primer and 4 units Ex‑Taq DNA polymerase [all Sangon Biotech (100% match) and the alignment data was classified into ten (Shanghai) Co., Ltd.]. The primer sequences are described in groups based upon the percentage of red line. Subsequently, Table I. The amplified products were subsequently cloned into the primers of the 148 CGIs (11) were aligned in sequence in a pUC18‑T vector [Sangon Biotech (Shanghai) Co., Ltd.] and the BLAST BGI YH database (CGI no. 103 was investigated sequenced using the Applied Biosystem 3730 DNA Analyzer with bisulfite sequencing as it lacked HpaII and HhaI sites (Life Technologies, Carlsbad, CA, USA). The results were and could not detected by HM-PCR). When a base differed further analyzed using the BDPC DNA methylation analysis to a sequence in the database, the primer was a 0% match. platform (http://services.ibc.uni-stuttgart.de/BDPC/BISMA/). The alignment results were divided into three groups: Perfect match (a pair of primers was 100% matched); 50% match (one Statistical analysis. Statistical analyses were performed primer of a pair was 100% matched); and no match (a pair of using SPSS 10.0 software (SPSS Inc., Chicago, IL, USA). primers was 0% matched). Comparisons between two groups were made using χ2 tests. P<0.05 was considered to indicate a statistically significant Study subjects and diagnosis. A total of 150 control cases and difference. 150 DS cases were obtained through the Children's Hospital of Chongqing Medical University (Chongqing, China) and Results participant's families or correspondents provided informed consent. The distribution of age and residential placement did CGI sequence alignment data from the BGI YH database. not differ between the control and the patients. Confirmation There are a total of 149 CGIs in chromosome 21q, according to of trisomy 21 was obtained by G‑banded karyotypes, all criteria outlined by Yamada et al (11). The alignment data from patients had complete trisomy 21, with 100% concordance the BGI YH database showed that 87% of CGIs (130 of 149) between cytogenetics and the clinical diagnosis of DS. Data were >80% matched; 13.42% were a perfect match (20 of 149), for Japanese individuals were obtained from the study by 22.14% were >95% matched (33 of 149), 21.48% were >90% Yamada et al (11). matched (32 of 149), 16.11% were >85% matched (24 of 149) and 14.09% were >85% matched (21 of 149). A match of Preparation of genomic DNA from human PBLs. Trisomy 21 <80% accounted for 13% of total CGIs (19 of 149); 2.01% and normal human lymphocytes were prepared from periph- were >75% matched (3 of 149), 8.05% were >70% matched eral blood. Total genomic DNA was extracted by TIANamp (12 of 149), 0.67% were >65% matched (1 of 149), 1.34% were Blood DNA kit (Tiangen Biotech, Beijing, China) following the >60% matched (2 of 149) and 0.67% were >55% matched manufacturer's instructions. The concentration of DNA was (1 of 149). The primers of the 148 CGIs were obtained from determined using the GeneQuant pro RNA/DNA Calculator the experimental design of Yamada et al (11). The alignment (GE Healthcare, Pittsburgh, PA, USA) and the integrity of data indicated that 98% (145 of 148) of the primers were a DNA was determined by electrophoresis. perfect match, two pairs of primers (CGI nos. 27 and 75) were a 50% match and one pair of primers (CGI no. 90) was not a HpaII‑McrBC PCR assay. Human genomic DNA (0.5 µg) match. New primers for CGI nos. 27, 75 and 90 were designed was digested with 15 units HpaII or HhaI (Promega Corp., using Primer 3 (v0.4.0; http://primer3.wi.mit.edu/) as follows: MOLECULAR MEDICINE REPORTS 9: 1681-1688, 2014 1683 1 isoform (PCNT) (ADAMTS5) transporter 5 CGI‑linked genes precursor ADAM metallopeptidase ADAM with thrombospondin type 1 Regulator of calcineurin 1 Runt ‑ related transcription PR domain zinc finger 15 (PRDM15) ES1 protein homolog, 3 (PCBP3) Protein C21orf2 isoform 4 3 Folate transporter 1 (rC) ‑ binding isoform 3 Folate isoform Poly protein Pericentrin motif (RCAN1) factor 1 (RUNX1) mitochondrial Ib (C21orf2) Bisulfite genomic sequencing primers ‑ 3' 5' ‑ GTAGATYGTGTAATTTTATTTATTAGTTAG ‑ 3' 5' ‑ CAAAACTACRAAATTCCCAAAC ‑ 3' 5' ‑ GAGTTTGTGGGATTTTGTAGTGAGT ‑ 3' 5' ‑ GGAGGTYGTTTTAGAAAGTTGAGA 5'‑GGGATAAYGATATTTTTTGGGG‑3' ‑ 3' 5' ‑ CCATACCRACTATTCTTTATTACATTC ‑ 3' 5' ‑ GTAGTTGGGATTATAGTTATATGTTATTATG ‑ 3' 5' ‑ GATGGTTTYGYGGGGTTAGG ‑ 3' 5' ‑ GTTTGAGGTTGGTTTAGGTTTTG ‑ 3' 5' ‑ CATCTCCCRAATATAAAACTTACTCC ‑ 3' 5' ‑ TATGGTGGTAGGTAAGAGAGTATGTG ‑ 3' 5' ‑ CCCRCAAACCCTAAATCTTAAC ‑ 3' 5' ‑ AAGATTTTGTAGTTGTAAGTGGTGTAG ‑ 3' 5' ‑ CCAACTAAATACATATTCTTCCTTCTC ‑ 3' 5' ‑ GATATTTTATATTTGTAGGGTTAGTGGA ‑ 3' 5' ‑ CAAAACCCCAATCAATCACAC ‑ 3' 5' ‑ CAACCCCAACTTCCTCTACTCC ‑ 3' 5' ‑ CCTCTAAATCCTTATCCCAAAAC ‑ 3' 5' ‑ RCCCTACAACAACACCRAAACC Japanese Unmethylation Unmethylation Unmethylation Complete methylation Unmethylation Complete methylation Complete methylation Complete methylation Complete methylation Methylation using HM ‑ PCR Chinese Unmethylation Unmethylation Incomplete methylation Unmethylation Incomplete methylation Incomplete methylation Complete methylation Incomplete methylation Complete methylation ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑

Japanese Composite methylation genomic sequencing Methylation using bisulfite Chinese ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ----- ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Unmethylation Unmethylation Incomplete methylation Unmethylation Complete methylation Incomplete methylation Incomplete methylation Complete methylation Incomplete methylation Complete methylation Table I. CGI DNA methylation statuses as determined by bisulfite genomic sequencing. I. CGI DNA Table CGI no. 14 ‑ 3' 5' ‑ CATCRCCTATCACCTAAACCC 38 41 75 103 109 129 133 134 46 CGI, CPG islands; HM-PCR, heavy methyl ‑ polymerase chain reaction. 1684 XIA et al: METHYLATION STATUS OF CpG ISLANDS ON CHROMOSOME 21q CGI ‑ linked genes ‑ 1(VI) chain precursor (COL6A1) α finger 2 (LONRF2) ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS1) PR domain zinc finger protein 15 (PRDM15) LON peptidase N ‑ terminal domain and ring Runt ‑ related transcription factor 1 (RUNX1) Holocarboxylase synthetase (HLCS) rich basic protein (WRB) Tryptophan Protein C21orf2 isoform 4 Folate transporter 1 isoform 3 (SLC19A3) Poly (rC) ‑ binding protein 3 (PCBP3) Collagen

Japanese

Composite methylation Complete methylation Figure 1. Human genomic DNA incubated with McrBC, HpaⅡ and HhaⅠ enzymes. Lanes 1, human genomic DNA; 2, DNA incubated with McrBC; 3, DNA incubated with HpaⅡ; and 4, DNA incubated with HhaⅠ.

Forward: 5'‑CTCTCACCGCCGCAAGTCGGTCGC‑3' and bisulfite genomic sequencing reverse: 5'‑CGTTGTTGGGGAACTTTTACTGTG‑3' for CGI Methylation status detected using no. 27; forward: 5'‑CAGCTCAAGAATGCACTGCATTT‑3' Chinese and reverse: 5'‑AGTCAAAACCCGGCTGGATTTCC‑3' for CGI no. 75; and forward: 5'‑GTATGTGCCACCAAA ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ------‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ TGATTATTCCT‑3' and reverse: 5'‑ACTCACTCTCCTAAC Incomplete methylation Unmethylation Incomplete methylation Incomplete methylation Incomplete methylation TTGAAGTTTTC‑3' for CGI no. 90.

HpaII‑McrBC PCR assay to evaluate 21q CGI allelic methyla‑ tion status. The electrophoresed images of genomic DNA and DNA products digested by McrBC, HpaⅡ or HhaⅠ enzymes, respectively, are shown in Fig. 1. When the genomic DNA were completely digested by the enzyme, the DNA products appeared as smeared bands in gel electrophoresis. Using the heavy Japanese methyl‑polymerase chain reaction (HM‑PCR) assay, results

showed that there was almost no difference in the DNA meth- Complete methylation Unmethylation Unmethylation Composite methylation Incomplete methylation Complete methylation Unmethylation Complete methylation Complete methylation Complete methylation ylation status of 21q CGIs among individuals with trisomy 21 and the control, as shown in Table II. A total of 148 CGIs in 21q were screened, including 102 null methylation, 26 complete

methylation, 7 composite methylation and 13 incomplete meth- Methylation status

ylation. However, 3 null methylation CGIs (nos. 12, 41 and 109), detected using HM‑PCR 2 complete methylation (nos. 129 and 134) and 1 composite methylation (no. 55) in Japanese patients were all incomplete

methylation in Chinese patients. In addition, 1 incomplete meth- Chinese ylation CGI (no. 68) and 1 complete methylation CGI (no. 75) in

Japanese patients were also incomplete methylation in Chinese ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ----- ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Composite methylation Incomplete methylation Incomplete methylation Incomplete methylation Unmethylation Unmethylation Incomplete methylation Incomplete methylation Incomplete methylation Composite methylation patients. Finally, 1 complete methylation CGIs (nos. 1 and 137) in Japanese patients were composite methylation in Chinese. In total, there were 10 CGIs that showed varying DNA methylation statuses among Japanese and Chinese patients, as presented in Fig. 2 and Table II. Table II. Variations in DNA methylation statuses between Chinese and Japanese patients determined using HM ‑ PCR. in DNA Variations II. Table CGI no. 1 12 41 55 68 75 109 129 134 137 HM-PCR, heavy methyl ‑ polymerase chain reaction. MOLECULAR MEDICINE REPORTS 9: 1681-1688, 2014 1685

Figure 2. Representative agarose gel showing polymerase chain reaction (PCR) products of ten methylation CGIs on chromosome 21q. Lane 1, marker; 2, PCR products of HpaⅡ digested DNA from a individual with Down's syndrome; 3, PCR products of McrBC digested DNA from a individual with Down's syndrome; 4, PCR products of HpaⅡ digested DNA from the control; and 5, PCR products of McrBC digested DNA from the control. CGI, CpG island.

Bisulfite genomic sequencing to confirm allelic methylation was detected by bisulfite sequencing, as it was short of the status. Nine CGIs (nos. 14, 38, 41, 75, 109, 129, 133, 134 and HpaII and HhaI recognition sites. The composite methylation 146) were selected to validate the HM‑PCR assay data using CGI no. 103 in Japanese patients was complete methylation in bisulfite sequencing in trisomy 21 and the control, as shown Chinese patients, as shown in Table I and Fig. 3. Furthermore, in Table I. These validations were determined with bisulfite the various methylation statuses of CpG dinucleotides in sequencing, which showed that CGI nos. 41, 109, 129 and 134 5 CGIs between trisomy 21 and the control were found, were incomplete methylation, CGI nos. 14, 38 and 75 were null including 5 sites in CGI no. 14, 6 sites in CGI no. 75, 14 sites in methylation and CGI nos. 133 and 146 were complete methyla- CGI no. 109, 1 site in CGI no. 134 and 3 sites in CGI no. 146. tion in Chinese patients, as shown in Fig. 3; these data validate A total of 29 CpG dinucleotides presented allele‑specific DNA the HM‑PCR results. The methylation status of CGI no. 103 methylation, as shown in Fig. 3. 1686 XIA et al: METHYLATION STATUS OF CpG ISLANDS ON CHROMOSOME 21q

Figure 3. Methylation status of ten CGIs on chromosome 21q as determined by bisulfite genomic sequencing, defined as methylated (red), unmethylated (blue) and unknown (white). The different methylation statuses in CpG dinucleotide sites between trisomy 21 and the controls are indicated by downward arrows. Image depicts the various methylation CpG dinucleotide sites in CGI no. 14 (nos. 3, 7, 11, 14 and 16), 75 (nos. 8, 18, 24, 25, 31 and 33), 109 (nos. 1, 3, 6, 10, 11, 12, 14, 15, 18, 19, 23, 25, 26 and 27), 134 (no. 1) and 146 (no. 1, 4 and 12). CGI, CpG island.

Discussion genes responsible for the CHD observed in DS (15‑17). Altered DNA methylation in 21q may be constitutively silenced over- Individuals with DS have an additional chromosome 21, which expressed genes in DS (10). Noteworthy gene candidates for is associated with the gene‑dosage effect and a wide spec- specific dysfunctions in DS are already emerging from these trum of phenotypic consequences, including life‑threatening research data. In the present study, CGI no. 75 was linked to complications, clinically significant alteration of life course PRDM15, which is a candidate gene for a particular pheno- (e.g., mental retardation) and dysmorphic physical features (12). type of DS or bipolar affective disorder (18). CGI no. 14 was The mechanisms of gene regulation, include function of associated with ADAMTS5, which is a protease involved in conserved nongenic regions, microRNA activities, RNA regulating aggrecanase activity in cartilage; deletion of active editing and DNA methylation. DS with a CHD is associated ADAMTS5 prevents cartilage degradation (19,20). CGI with a global hypomethylation status (13). DNA methylation no. 109 was associated with C21orf2, which is involved in is a possible mechanism of alteration, which the regulation of cell morphology and cytoskeletal organiza- may contribute to various abnormalities. Chango et al (14) tion (21). CGI no. 134 was associated with PCBP3, which is used a combination of methylation‑sensitive arbitrarily associated with frontotemporal dementia and hypothesized primed PCR and quantitation of DNA fragments to find six to participate in mRNA metabolic processes (22,23). CGI fragments that were hypermethylated in PBLs from eight indi- no. 146 was associated with PCNT, which may be important in viduals with DS, compared with eight normal controls. Kerkel preventing premature centrosome splitting during interphase et al (10) observed that 8 genes had different methylation by inhibition of NEK2 kinase activity at the centrosome (24). status between the DS patients and normal controls. One of The methylation alteration may be associated with the DS the 8 genes is named SUMO3 and is located on chromosome phenotype, as insights from investigating DS as a model 21. The current observations are consistent with this data. system have shed light on potential epigenetic biomarkers for There were differences in the DNA methylation status of CpG noninvasive prenatal diagnosis in the general population. dinucleotide sites in 21q CGIs (nos. 14, 75, 109, 134 and 146) The fetal‑specific DNA methylation ratio permits noninva- among individuals with trisomy 21 and the control. Molecular sive prenatal diagnosis of trisomy 21 by analyzing fetal‑specific analysis reveals that the 21q22.1‑q22.3 region, also known as DMRs in free fetal DNA of the maternal circulation during the DS critical region (DSCR), appears to contain the gene or pregnancy (25‑28). A few differentially methylated sequences MOLECULAR MEDICINE REPORTS 9: 1681-1688, 2014 1687 are fetal DNA methylation markers, including HLCS and distinguishing ethnicities. The different DNA methylation DSCR4 located at chromosome 21, RASSF1A located at status of CpG dinucleotides between individuals with DS and chromosome 3 and ZFY located at chromosome Y (29‑31). the control may contribute to the DS complex phenotypes and However, these markers were not specific for individuals with be a potential epigenetic marker for diagnosing trisomy 21. DS. In the present study, methylation alteration in DS may represent a specific epigenetic biomarker for future prenatal Acknowledgements diagnosis; however, DNA methylation must be further verified in fetuses with DS. The authors would like to thank Dr Lin Zou and Dr Jing Yang DNA methylation in DS individuals did not change signifi- (Center for Clinical Molecular Medicine, Children's Hospital cantly in comparison with the controls (14). In the present of Chongqing Medical University) for case collection. The study, there was no significant difference in 21q CGIs in present study was supported by grants from the Chongqing Chinese patients, as determined using HM‑PCR; this result Key Laboratory of Birth Defects and Reproductive Health was in accordance with observations from Kerkel et al (10) (grant no. 0901) and the Natural Science Foundation Project of no significant difference between normal and DS samples. of CQ CSTC (grant no. 2009BA5082). While the different methylation statuses of CpG dinucleotide sites between the normal and DS do not cause the difference in References the CGIs' methylation status as screened by HM PCR assays, 1. Huether CA, Gummere GR, Hook EB, et al: Down's syndrome: because CGIs comprise numerous CpG dinucleotide sites only percentage reporting on birth certificates and single year maternal a few altered methylation levels are likely to not affect HM PCR age risk rates for Ohio 1970‑79: comparison with upstate New results (11). 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